Biomolecular arrays have quickly developed into an important tool in life science research. Microarrays, or densely-packed, ordered arrangements of miniature reaction sites on a suitable substrate, enable the rapid evaluation of complex biomolecular interactions. Because of their high-throughput characteristics and low-volume reagent and sample requirements, microarrays are now commonly used in gene expression studies, and they are finding their way into significant emerging areas such as proteomics and diagnostics.
The reaction sites of the array can be produced by transferring to the substrate droplets containing biological or biochemical material. A variety of techniques can be used, including contact spotting, non-contact spotting, and dispensing. With contact spotting, a fluid bearing pin leaves a drop on the surface when the pin is forced to contact the substrate. With non-contact spotting, a drop is pulled from its source when the drop touches the substrate. With dispensing, a drop is delivered to the substrate from a distance, similar to an inkjet printer. Reaction sites on the array can also be produced by photolithographic techniques (such as those employed by Affymetrix or NimbleGen, for example).
The quality of the reaction sites directly affects the reliability of the resultant data. Ideally, each site would have a consistent and uniform morphology and would be non-interacting with adjacent sites, so that when a reaction occurred at a given site, a clear and detectable response would emanate from only that one site, and not from neighboring sites or from the substrate. To reduce the overall size of an array while maximizing the number of reaction sites and minimizing the required reagent and sample volumes, the sites on the array should have the highest possible areal density.
With current microarray technology, which is dominated by the use of flat substrates (often glass microscope slides), areal density is limited. To increase the signal from a given reaction site, the interaction area between the fluid (usually aqueous) and the substrate should be maximized. One way to do this is by using a surface that promotes wetting. A flat surface that promotes wetting, however, can lead to spots (and thus reaction sites) having irregular shapes and compositions. A flat wetting surface can also lead to the spreading of fluid from its intended site into neighboring sites. Thus, flat surfaces are intrinsically limited by fluid-surface interactions that force a tradeoff between the desired properties of the reaction sites.
To make the sites more uniform, the surface can be made non-wetting. Unfortunately, this reduces the interaction area between the fluid and the surface, thereby reducing the signal that would otherwise be obtainable. In addition, since droplets do not adhere well to a flat non-wetting surface, deposition volumes can vary from site to site, and droplets can slide away from their intended location, unless they are otherwise confined.
One way of avoiding the wetting vs. non-wetting dichotomy is to prepare surfaces that have regions of varying hydrophilic/hydrophobic contrast. Due to the aqueous environment of biomolecular arrays, patterned media having hydrophilic/hydrophobic contrast are ideal for confining bioactivity to within discrete regions defined by the pattern, with each discrete region in effect acting as an individual bio-probe. A hydrophobic surface is generally regarded as one having a static water contact angle of greater than 90 degrees, with decreasing contact angles resulting in progressively more hydrophilic surfaces. A surface having a water contact angle of less than 65 degrees is considered strongly hydrophilic. (For a discussion of contact angles, see A. W. Adamson et al., “Physical chemistry of surfaces”, John and Wiley & Sons, New York, 1997.)
Several methods have been reported for preparing patterns of varying hydrophilicity, including traditional lithographic methods, imprinting, and contact printing. Lithographic techniques rely on the attachment of hydrophobic (or hydrophilic) molecules to preselected regions defined by photoresists in a hydrophilic (or hydrophobic) matrix. (See, for example, J. H. Butler et al., J. Am. Chem. Soc. 2001, 123, 8887.) With imprinting techniques, hydrophilic regions are created by pipetting droplets of a washable or hydrophilic lacquer, much like that in an ink-jet printer, and then converting the adjacent regions to hydrophobic regions. (See, for example, UK Patent Application GB 2340298AUK and Patent Application GB 2332273A.) Contact printing methods typically involve elastomeric stamps with hydrophilic (or hydrophobic) inks, with hydrophilic (or hydrophobic) patterns being generated as a result of transferring the ink onto a substrate. (See, for example, G. MacBeath et al, Science 2000, 289, 1760; and C. M. Niemeyer et al., Angew. Chem. Int. Ed. 1999, 38, 2865). U.S. Pat. No. 5,939,314 to Koontz discloses porous polymeric membranes having hydrophilic/hydrophobic contrast, in which the pore size is on the order of 0.1–2000 microns, but pores of this size are still relatively large. These methods generally involve, however, a series of several process steps.
A simple, more effective route to patterned substrate arrays having regions of varying hydrophilic/hydrophobic contrast would be highly desirable. Further, such arrays should have a high areal density of sites and high effective surface area to permit the collection of data with good signal/noise ratio. In addition, such an apparatus would ideally have sites of consistent and uniform spot morphology.